Feed media

ABSTRACT

The invention provides stable feed media containing pyruvate and methods for stabilizing feed media by adding pyruvate. The invention further provides methods for producing proteins using such media and proteins produced through the use of such methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/917,569, filed May 11, 2007, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention is in the field of cell culture media and methods.

BACKGROUND OF THE INVENTION

Large-scale fed-batch culture of mammalian cells, especially ChineseHamster Ovary (CHO) cells, is widely used to produce proteins used in avariety of applications, such as diagnostic, therapeutic, and researchuses. Particular attention has focused on CHO cells because they havebeen extensively characterized and have been approved for use inclinical manufacturing by regulatory agencies. Such cultures aretypically maintained for days, or even weeks, while the cells producethe desired protein(s). During this time the culture can be supplementedwith a feed medium containing components, such as nutrients and aminoacids, which are consumed during the course of the culture. Such feedinghas been shown to improve protein production by a mammalian cellculture. See e.g., U.S. Pat. No. 5,672,502. Even incrementalimprovements in protein production can be valuable, given the expenseand difficulty of building and obtaining regulatory approval forlarge-scale, commercial culture facilities.

Concentrated feed media are often used in fed batch culture processes toimprove protein titer, cell growth, and/or cell viability. Somecomponents present at high concentration in feed media may precipitateduring storage, especially when the pH of the medium is near neutrality.Precipitation of medium components during storage prior to use of amedium is very undesirable because it adds an element of uncertainty.When medium components precipitate, the concentration of mediumcomponents in solution, versus in the precipitate, will be unknown.Since concentrations of various medium components can affect thequantity and quality of a protein produced by a culture, this is anelement of uncertainty that is highly undesirable in a commercialculture process, in which culture conditions are, optimally, carefullycontrolled. Moreover, commercial processes may be subjected to stringentregulatory review. Thus, feed media with high concentrations of aminoacids that can be stored for a period of time without precipitatingwould provide significant advantages.

SUMMARY OF THE INVENTION

The invention provides stable feed media containing pyruvate, methodsfor stabilizing feed media comprising adding pyruvate to a medium,methods for using stable feed media, and proteins produced by culturesfed with a medium of the invention.

In one embodiment, the invention encompasses a method for stabilizing aconcentrated feed medium to be used for feeding a mammalian cell culturecomprising adding to the feed medium at least about 9, 18, 25, 30, 35,40, 45, or 50 mM pyruvate, wherein the feed medium can comprise cysteineand/or cystine, wherein the sum of the concentrations of cysteine and/orcystine can be at least about 7.9 mM, wherein the pH of the feed mediumcan be from about 5.8 to about 7.4, wherein the feed medium can comprisetyrosine at a concentration of not more than about 4.4 mM or 4.6 mMtyrosine, and wherein the medium can be stable for at least about 1, 2,or 3 weeks at room temperature. The pyruvate can be sodium pyruvate. ThepH of the feed medium can be from about 6.0 to about 7.2 and/or at leastabout 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8 and not more than about 7.4. Thefeed medium can comprise at least about 5.0, 6.0, 7.0, 12.0, 21.0, 35.0,40.0 or 45.0 mM cysteine and/or at least about 0.5, 1.0, 1.5, 2.0, or4.0 mM cystine. The feed medium may comprise from about 7 mM to about 16mM cysteine or from about 7.5 mM to about 13 mM cysteine. The feedmedium can comprise tyrosine at a concentration of at least about 2, 3,or 4 mM tyrosine. The feed medium can comprise a protein hydrolysate andmay be serum free. The osmolarity of the feed medium can be from about200 mOsm to about 1300 mOsm, from about 250 mOsm to about 1000 mOsm,from about 200 mOsm to about 500 mOsm, from about 500 mOsm to about 1000mOsm, from about 700 mOsm to about 900 mOsm, from about 270 mOsm toabout 900 mOsm, from about 300 mOsm to about 830 mOsm, or from about 200mOsm to about 400 mOsm. The mammalian cell culture can contain CHOcells.

In another embodiment, the invention provides a method for stabilizing afeed medium comprising adding about 30 to 40 mM pyruvate to a feedmedium which can comprise (a) from about 3 mM and to about 4.0 mMtyrosine, and (b) cysteine and/or cystine, wherein the sum of theconcentrations of cysteine and/or cystine is at least about 7.9 mM, andwherein the pH of the feed medium is from about 5.8 to about 7.4. The pHmay be from about 6.0 mM to about 7.4 mM and/or at least about 6.3, 6.4,6.5, 6.6, 6.7, or 6.8 and not more than about 7.4. The feed medium canbe stable for at least about 1, 2, 3, or 4 weeks at room temperature orat 4-8° C.

In a further embodiment, the invention comprises a feed medium for amammalian cell culture that can comprise at least about 9, 18, 25, 30,35, 40, 45, or 50 mM pyruvate and at least about 5 mM cysteine, whereinthe pH of the feed medium can be from about 5.8 to about 7.4, whereinthe feed medium can comprise tyrosine at a concentration of not morethan about 4.4 mM, and wherein the medium can be stable for at leastabout 1, 2, or 3 weeks at room temperature or at 4-8° C. The pyruvatecan be sodium pyruvate. The pH of the feed medium can be from about 6.0to about 7.2 and/or at least about 6.3, 6.4, 6.5, 6.6, 6.7, or 6.8 andnot more than about 7.4. The feed medium can also comprise at leastabout 6, 7, 12, 21, 35, 40, or 45 mM cysteine and/or at least about 0.5,1.0, 1.5, 2.0, or 4.0 mM cystine. The feed medium can comprise tyrosine,optionally at a concentration of at least about 2, 3, 4, or 4.2 mMtyrosine. The feed medium can comprise a protein hydrolysate and may beserum free. The osmolarity of the feed medium can be from about 200 mOsmto about 1300 mOsm, from about 250 mOsm to about 1000 mOsm, from about200 mOsm to about 500 mOsm, from about 500 mOsm to about 1000 mOsm, fromabout 700 mOsm to about 900 mOsm, from about 270 mOsm to about 900 mOsm,from about 300 mOsm to about 830 mOsm, or from about 200 mOsm to about400 mOsm. The mammalian cell culture can be a Chinese Hamster Ovary(CHO) cell culture.

In a further embodiment, the invention encompasses a feed medium for aCHO cell culture, which can comprise (1) from about 3 mM and to about4.0 mM tyrosine, (2) cysteine and/or cystine, wherein the sum of theconcentrations of cysteine and/or cystine is at least about 7.9 mM, and(3) about 30 to 40 mM pyruvate, wherein the pH of the feed medium can befrom about 5.8 to about 7.4 or from about 6.0 to about 7.4. The feedmedium can be stable for at least about 1, 2, 3, or 4 weeks at roomtemperature or a 4-8° C.

In other embodiments, the invention provides various methods ofutilizing the feed media of the invention. For example, the inventionprovides a method for culturing cells comprising culturing the cells ina base medium and feeding the culture with a feed medium of theinvention. Further, the invention encompasses a method for producing aprotein comprising culturing mammalian cells that produce the protein ina base medium, feeding the culture with a feed medium of the invention,and recovering the protein from the culture medium. The protein may be arecombinant protein and can be purified. The base medium used in theculture may be serum free, and the cells may be cultured in at least onegrowth phase and at least one production phase. In still another aspect,the invention provides a method for increasing production of a proteinproduced by cultured mammalian cells, which may be CHO cells, comprisingfeeding the cultured cells with a feed medium of the invention. Feedingmay occur one or more times during the culture and may be adjusted so asto keep certain culture components within certain concentration ranges.

In a further aspect, the invention encompasses a protein produced by anyof the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Protein titers at 8 (left) or 10 (right) days of culture areindicated by the bars. The cells are CHO cells producing a recombinantprotein. The cells were cultured as described in Example 1 and fed witha feed media as described in Example 1 and Table 2.

FIG. 2A: This figure was created using the day 21 data described inExample 4 and Table 5 and using JMP® software (SAS Institute Inc., Cary,N.C.). The upper row of five boxes show the likelihood of having noprecipitate “P(day 21=0),” and the lower row of five boxes show thelikelihood of having a precipitate “P(day 21=1)” at day 21. On thevertical y axes, 1.00 means 100% probability, and 0.00 means zeroprobability. The dotted vertical lines in each box indicate theconcentration at which the medium component or storage temperaturelisted below each vertical column of two boxes is set in all othercolumns of boxes. In the column of two boxes directly over the labeledcomponent or temperature, the concentration of the component or thetemperature is varied in the range shown along the x axis below eachvertical column of two boxes. The concentrations listed along the x axisare expressed in millimolar units, and the temperatures are expressed asdegrees centigrade. For example sodium pyruvate is set at 34.65 mM inall boxes other than the two directly above the words “sodium pyruvate.”In these two boxes, sodium pyruvate varies from about 5 mM to almost 35mM.

FIG. 2B: This figure is the same as FIG. 2A except that sodium pyruvateis set at 4.9 mM in all columns except that labeled “sodium pyruvate.”

FIG. 3A: This figure was made using the day 21 data described in Example5 and Table 6 using JMP® software (SAS Institute Inc., Cary, N.C.). Itis similar to FIG. 2A, although the exact concentrations at which thevarious medium components are set (marked directly over the name of themedium component) differs somewhat. Sodium pyruvate is set at 35.11 mMin all columns other than the rightmost column, in which the sodiumpyruvate concentration varies.

FIG. 3B: This figure is like FIG. 3A except sodium pyruvate is set at aconcentration of 4.54 mM in all columns other than the rightmost columnin which the concentration of sodium pyruvate varies.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides concentrated feed media for use in fedbatch culture of eukaryotic, optionally mammalian, cells that can besoluble at room temperature and can be stored for a reasonable timewithout precipitation. For use in mammalian cell culture, a feed mediumcan have a pH such that, when added to the culture, it will not bringthe culture outside of a physiologic range, for example, from about pH6.5 to about 7.5. These stable, concentrated feed media can contain highconcentrations of amino acids such as cysteine and/or cystine and/ortyrosine and high concentrations of pyruvate. The invention thuscontributes to more operationally advantageous and robust cell cultureprocesses. The feed media of the invention are particularly useful forlarge scale, commercial cultures of mammalian cells that produce arecombinant protein, which can be used as, for example, a therapeutic, adiagnostic, or a research reagent. The feed medium of the invention maybe stored at room temperature or at refrigerator temperature.

The term “stable,” as used herein, refers to a medium that does notprecipitate upon storage for at least a specified period of time, suchas at least about 1 week, 2 weeks, 3 weeks, or 4 weeks. The medium maybe stored, for example, at room temperature (which is 15-30° C., asmeant herein) or at refrigerator temperature (which is 4-8° C., as meantherein). Similarly, when a medium is said to be “stabilized” for someperiod of time, it means that solutes in the media do not form aprecipitate and come out of solution.

“Pyruvate” includes the free form of pyruvic acid as well as acid salts,including sodium pyruvate and other acid salts.

A “base medium,” as meant herein, is a medium used for culturingeukaryotic cells which is, itself, directly used to culture the cellsand is not used as an additive to other media, although variouscomponents may be added to a base medium. For example, if CHO cells werecultured in DMEM, a well-known, commercially-available medium formammalian cells, and periodically fed with glucose or other nutrients,DMEM would be considered the base medium.

A “feed medium” is a medium used as a feed in a culture of eukaryoticcells, which may be mammalian cells. A feed medium, like a base medium,is designed with regard to the needs of the particular cells beingcultured. Thus, a base medium can be used as a basis for designing afeed medium. As described below in more detail, a feed medium can havehigher concentrations of most, but not all, components of a base culturemedium. For example, some components, such as, for example, nutrientsincluding amino acids or carbohydrates, may be at about 5×, 6×, 7×, 8×,9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, oreven about 1000× of their normal concentrations in a base medium. Somecomponents, such as salts, may be kept at about 1× of the base mediumconcentration, as one would want to keep the feed isotonic with the basemedium. Components not normally utilized as nutrients in media would notgenerally be present at increased concentrations in feed media. Thus,some components are added to keep the feed physiologic, and some areadded because they are replenishing nutrients to the culture.

A “recombinant protein” is a protein resulting from the process ofgenetic engineering. Cells have been “genetically engineered” to expressa specific protein when recombinant nucleic acid sequences that allowexpression of the protein have been introduced into the cells usingmethods of “genetic engineering,” such as viral infection with arecombinant virus, transfection, transformation, or electroporation. Seee.g. Kaufman et al. (1990), Meth. Enzymol. 185: 487-511; CurrentProtocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, NewYork, 1988, and quarterly updates). The term “genetic engineering”refers to a recombinant DNA or RNA method used to create a host cellthat expresses a gene at elevated levels or at lowered levels, orexpresses a mutant form of the gene. In other words, the cell has beentransfected, transformed or transduced with a recombinant polynucleotidemolecule, and thereby altered so as to cause the cell to alterexpression of a desired protein. The methods of “genetic engineering”also encompass numerous methods including, but not limited to,amplifying nucleic acids using polymerase chain reaction, assemblingrecombinant DNA molecules by cloning them in Escherichia coli,restriction enzyme digestion of nucleic acids, ligation of nucleicacids, and transfer of bases to the ends of nucleic acids, amongnumerous other methods that are well-known in the art. See e.g. Sambrooket al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3,Cold Spring Harbor Laboratory, 1989. Methods and vectors for geneticallyengineering cells and/or cell lines to express a protein of interest arewell known to those skilled in the art. Genetic engineering techniquesinclude but are not limited to expression vectors, targeted homologousrecombination and gene activation (see, for example, U.S. Pat. No.5,272,071 to Chappel) and trans activation by engineered transcriptionfactors (see e.g., Segal et al., 1999, Proc. Natl. Acad. Sci. USA96(6):2758-63). Optionally, the proteins are expressed under the controlof a heterologous control element such as, for example, a promoter thatdoes not in nature direct the production of that protein. For example,the promoter can be a strong viral promoter (e.g., CMV, SV40) thatdirects the expression of a mammalian protein. The host cell may or maynot normally produce the protein. For example, the host cell can be aCHO cell that has been genetically engineered to produce a humanprotein, meaning that nucleic acid encoding the human protein has beenintroduced into the CHO cell. Alternatively, the host cell can be ahuman cell that has been genetically engineered to produce increasedlevels of a human protein normally present only at very low levels(e.g., by replacing the endogenous promoter with a strong viralpromoter).

“Substantially similar” proteins are at least about 90% identical toeach other in amino acid sequence and maintain or alter in a desirablemanner the biological activity of the unaltered protein. As is known inthe art, changes in conserved amino acids are more likely to affect thebiological function of a protein. Further, conservative amino acidsubstitutions at any site in a protein are less likely to causefunctional changes than non-conservative substitutions. Conservativeamino acid substitutions, unlikely to affect biological activity,include, without limitation, the following: Ala for Ser, Val for Ile,Asp for Glu, Thr for Ser, Ala for Gly, Ala for Thr, Ser for Asn, Ala forVal, Ser for Gly, Tyr for Phe, Ala for Pro, Lys for Arg, Asp for Asn,Leu for Ile, Leu for Val, Ala for Glu, Asp for Gly, and these changes inthe reverse. See e.g. Neurath et al., The Proteins, Academic Press, NewYork (1979). In addition exchanges of amino acids among members of thefollowing six groups of amino acids will be considered to beconservative substitutions for the purposes of the invention. The groupsare: 1) methionine, alanine, valine, leucine, and isoleucine; 2)cysteine, serine, threonine, asparagine, and glutamine; 3) aspartate andglutamate; 4) histidine, lysine, and arginine; 5) glycine and proline;and 6) tryptophan, tyrosine, and phenylalanine. The percent identity oftwo amino sequences can be determined using the Genetics Computer Group(GCG; Madison, Wis.) Wisconsin package version 10.0 program, ‘GAP’(Devereux et al. (1984), Nucl. Acids Res. 12: 387) or other comparablecomputer programs. The preferred default parameters for the ‘GAP’program includes: (1) the weighted amino acid comparison matrix ofGribskov and Burgess (1986), Nucl. Acids Res. 14: 6745, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979), or othercomparable comparison matrices; (2) a penalty of 8 for each gap and anadditional penalty of 2 for each symbol in each gap for amino acidsequences; (3) no penalty for end gaps; and (4) no maximum penalty forlong gaps.

Fed batch culture is a widely-practiced culture method for large scaleproduction of proteins from mammalian cells. See e.g. Chu and Robinson(2001), Current Opin. Biotechnol. 12: 180-87. A fed batch culture ofmammalian cells is one in which the culture is fed, either continuouslyor periodically, with a feed medium containing nutrients. Feeding canoccur on a predetermined schedule of, for example, every day, once everytwo days, once every three days, etc. Alternatively or in addition, theculture can be monitored for specific medium components, for example,glucose and/or glutamine and/or any amino acid, and feedings can beadjusted so as to keep one or more of these components within a desiredrange. When compared to a batch culture, in which no feeding occurs, afed batch culture can produce greater amounts of protein. See e.g. U.S.Pat. No. 5,672,502.

A feed medium of the invention will generally contain nutrients that aredepleted during cell culture. A feed medium of the invention willtypically contain most of the components of a typical mammalian basecell culture medium, but with some components, such as those generallyviewed as nutrients, at high concentrations so as to avoid too muchdilution of the culture. Particularly in culture used for proteinproduction, it is advantageous to increase culture volume as little aspossible with media feeds so as to maximize the amount of proteinproduced per unit volume. At large scale, an increase of, for example,fifty percent in volume, can create significant handling issues. A feedmedium of the invention may contain many of amino acids found in aculture medium, but at, for example, about 5×, 6×, 7×, 8×, 9×, 10×, 12×,14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×, or even about1000× of their usual concentration in a base medium. The amino acids caninclude alanine, arginine, asparagine, aspartate, cysteine, cystine,glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine. As meant herein, such amino acids are the “L”stereoisomers commonly found in nature, rather than “D” stereoisomers,which are not commonly found in terrestrial living systems. For example,“cysteine” refers to L-cysteine rather than D-cysteine. Carbohydratessuch as glucose or mannose, galactose, fructose, sucrose, orglucosamine, etc. can also be added to a feed medium of the invention oradded a culture separately. Vitamins, proteins, serum, buffering agents,salts, and hydrolysates of soy, casein, and yeast may or may not be partof a feed medium of the invention. These feed media may be serum free.Feed media of the invention may or may not contain growth factors, suchas IGF-1 or insulin. Feed media of the invention can be protein freeand/or chemically defined, i.e., protein free, hydrolysate free, andserum free.

With regard to salts, such as, for example sodium chloride, theconcentration used in a feed medium of the invention can be calculatedsuch that the osmolarity of the culture does not go beyond an optimalrange of from about 270 mOsm to about 550 mOsm or from about 270 mOsm toabout 450 mOsm. In some embodiments, the osmolarity of the culture mayrange from about 250 mOsm to about to about 650 mOsm, or from about 260mOsm to about 600 mOsm. The feed medium itself can have a wider range ofosmolarity since it is diluted upon addition to the culture. Thus, afeed medium can have an osmolarity of from about 200 mOsm to about 1300mOsm, from about 250 mOsm to about 1000 mOsm, from about 500 mOsm toabout 1000 mOsm, from about 700 mOsm to about 900 mOsm, from about 270mOsm to about 900 mOsm, from about 300 mOsm to about 830 mOsm, fromabout 200 mOsm to about 500 mOsm, or from about 200 mOsm to about 400mOsm. Addition of a protein hydrolysate to a feed medium can contributeto higher osmolarity. Some salts may be omitted entirely from a feedmedium. Thus, it is generally nutrients that are consumed during cellculture (such as amino acids and carbohydrates), rather than salts,buffers, or shear protectants, that are present in high concentrationsin a feed medium.

Mammalian cells grown in culture generally can be cultured at nearneutral pHs, such as from about pH 6.5 to about pH 7.5. Thus, althoughfeed media of the invention can be somewhat outside this range, theaddition of the feed medium will preferably not bring the pH of theentire culture outside this range. Thus, feed media can have a pH fromabout 5.8 to about 8.0, or from about 6.0 to about 7.8, or from about6.1 to about 7.5, or from about 6.5 to about 7.4, from about 5.8 toabout 7.4, or from about 6.0 to about 7.2. In some embodiments, the pHof a feed medium can be about 6.8, 6.9, 7.0, 7.1, or 7.2.

Most commonly-used components of mammalian culture feed medium arefreely soluble in water. However, a few amino acids have limitedsolubility in water. For example L-cystine, an oxidized form of cysteineoften used in culture media, is soluble at a concentration of up to only0.112 g/L in water at 25° C., and L-tyrosine is soluble at aconcentration of up to only 0.045 g/100 g of water (equivalent to 0.45g/L) at 25° C. THE MERCK INDEX, 12^(th) Ed., Budavari et al., eds.,Merck & Co., Inc., 1996, p. 471 and 9971. Cysteine readily oxidizes toform cystine in neutral or slightly alkaline aqueous solutions. Ibid,pp. 470-71. Thus, even though cysteine, itself, is freely water soluble,it may contribute to insolubility and/or precipitation of a medium inits oxidized form, cystine. Tyrosine is soluble in alkaline solutions,and cystine is quite soluble in solutions below pH 2 or above pH 8.Ibid, p. 471 and 9971. Since a feed medium is generally close to neutralpH, consistent with the requirements of mammalian cells, even moderateconcentrations of tyrosine and/or cystine can present problems withmedium stability. Furthermore, since cysteine can be oxidized to cystinein a neutral solution in the presence of air, cysteine may causeprecipitation, even though cysteine, itself, is quite soluble in aqueoussolutions. Thus, there is a need in the art for methods to stabilizefeed media, which often contain high concentrations of amino acids suchas cystine, tyrosine, and cysteine and have approximately neutral pH.

The concentration of pyruvate used in the feed media and methods of theinvention can be at least about 0.9, 3, 5, 9, 10, 18, 20, 25, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 mM pyruvate and not more thanabout 40, 45, 50, 100, 200, or 315 mM pyruvate. Alternatively, the feedmedia of the invention can contain about 18, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 45 mMpyruvate. The pyruvate concentration of the feed media can range fromabout 20 mM to about 315 mM, from about 20 mM to about 200 mM, fromabout 20 mM to about 100 mM, from about 20 mM to about 50 mM, from about25 mM to about 45 mM, from about 25 mM to about 40 mM, or from about 30mM to about 40 mM. The pyruvate can be sodium pyruvate.

The concentration of cysteine in a feed medium of the invention can beat least about 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0,11.0, 12.0, 14.0, 16.0, 18.0, or 20.0 mM and/or not more than about 8.0,9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 18.0, 20.0, 25.0, 30.0,35.0, 40.0, 60.0 80.0, or 100.0 mM. Alternatively, the concentration ofcysteine added to a feed medium of the invention can be from about 3 mMto about 40 mM, from about 5 mM to about 35 mM, from about 7 mM to about30 mM, from about 8 mM to about 25 mM, or from about 7.5 mM to about 15mM, or about 8, 10, 12, 14, 16, 18, or 20 mM.

Cystine can be present in a feed medium of the invention at aconcentration from about 0.1 mM to about 2.5 mM, from about 0.5 mM toabout 1.5 mM, from about 1.0 mM to about 1.2 mM, or about 1 mM or 1.1mM. Alternatively, cystine can be added at a concentration of at leastabout 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, or 1.0 mM and/or not more than 0.4,0.6, 0.8, 1.0, 1.2, 1.4, 2, 3, 5, or 10 mM. Recognizing that some of thecysteine added to a medium may oxidize to form cystine or some of thecystine added to a medium may be reduced to form cysteine, the numbersgiven above for these concentrations refer to the concentration ofcysteine or cystine which is actually added to the medium without laterdetermination of what proportion of this may have been oxidized orreduced.

Either cysteine, cystine, or tyrosine may be omitted from a feed mediumof the invention. Tyrosine may be present at less than or equal to about4.6, 4.5, 4.4, 4.3, 4.2, or 4.1 mM. Tyrosine may be present in aconcentration of at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or4.0 mM. If present, tyrosine may be at a concentration greater thanabout 1 mM and less than about 4.4 or 4.6 mM, from about 2 mM to about4.4 mM, from about 3 mM to about 4.0 mM, from about 3 mM to about 4.4mM, or from about 4.0 mM to about 4.4 mM. Alternatively, tyrosine can bepresent in the feed media of the invention at about 3, 3.2, 3.4, 3.6,3.8, 3.9, 4.0, 4.2, 4.4, or 4.5 mM.

A feed medium of the invention contains pyruvate, which can stabilizethe medium. As explained above, a feed medium can also containcomponents such as cystine, cysteine, and tyrosine, which maydestabilize the medium. Other components that are relatively insolublein water may be included in a feed medium of the invention, providedthat they are at concentrations such that the medium is stable for atleast about 1, 2, or 3 weeks at room temperature. Some relativelyinsoluble components may form a separate, non-aqueous phase, and suchcomponents may or may not be included in a feed medium of the invention.

Table 1 (below) gives an exemplary list of the components that may beincluded in a feed medium of the invention and concentration ranges atwhich each component might be used. Depending on the needs of the cells,not all of these components need be present at all. Alternatively, acomponent may be present in a concentration outside of the ranges statedin Table 1. Moreover, components other than those listed in Table 1 canbe included in a feed medium of the invention. Such additional mediumcomponents may, for example, include alanine, aspartate, glutamate,phenol red, or various vitamins including Vitamins A, D2, or B12 orascorbic acid (Vitamin C) or alpha tocopherol phosphate, among manyothers.

TABLE 1 Composition of Exemplary Feed Media Feed Media ComponentConcentration Range (mM) L-Arginine  1.0-57.0 L-Asparagine   8.0-200.0Biotin (B7) 0.0003-0.05  Calcium Chloride 0.09-9.0 D-CalciumPantothenate 0.02-2.1 Choline Chloride 0.008-55.0 Cupric Sulfate0.00012-0.0012 Cyanocobalamin (B12) 0.01-0.6 L-Cysteine   2.1-105.0L-Cystine  0.3-5.0 Ferric Nitrate  0.7-7.5 Folic Acid 0.02-2.3 D-GlucoseFed on demand L-Glutamine   8.2-206.0 Glycine  1.2-80.0 L-Histidine 0.6-67.0 Hypoxanthine  0.3-19.0 i-Inositol  0.1-59.0 L-Isoleucine 1.6-80.0 L-Leucine   2.2-115.0 Linoleic Acid 0.003-0.04 DL-Alpha-LipoicAcid 0.01-1.0 L-Lysine   2.4-165.0 Magnesium Chloride 0.10-6.3 MagnesiumSulfate  0.81-12.5 L-Methionine  0.6-31.0 Niacinamide (B3) 0.05-2.5L-Phenylalanine  0.7-46.0 Potassium Chloride  4.0-40.0 PotassiumPhosphate monobasic  0.3-37.0 Potassium Phosphate dibasic  0.8-58.0L-Proline   2.6-130 Putrescine 0.019-0.53 Pyridoxal, HCl 0.02-2.2Pyridoxine, HCl 0.0005-0.2  Riboflavin (B2) 0.002-0.16 L-Serine  1.1-143.0 Sodium bicarbonate    0-30.0 Sodium Chloride As neededSodium Phos Dibas Anhy  0.4-71.0 Sodium Phos Monobas  0.3-66.0 SodiumPyruvate   3.0-454.0 Thiamine (B1) 0.003-1.4  L-Threonine   2.5-126.0Thymidine 0.04-1.9 L-Tryptophan  0.2-11.0 L-Tyrosine  0.3-4.6 L-Valine  2.5-128.0 Zinc Sulfate 0.03-1.6

Proteins to be expressed by the cultured cells can be protein-baseddrugs, also known as biologics. The proteins can be secreted asextracellular products. The protein being produced can comprise part orall of a protein that is identical or substantially similar to anaturally-occurring protein, and/or it may, or may not, be a recombinantfusion protein. Optionally, the protein may be a human protein, afragment thereof, or a substantially similar protein that is at least 15amino acids in length. It may comprise a non-antibody protein and/or anantibody. It may be produced intracellularly or be secreted into theculture medium from which it can be recovered. It may or may not be asoluble protein.

The protein being produced can comprise part or all of a protein that isidentical or substantially similar to a naturally-occurring protein,and/or it may, or may not, be a recombinant fusion protein. Arecombinant fusion protein is a polypeptide that is a fusion of part ofall of two different proteins. For example, the extracellular region ofa receptor fused to the Fc region of an antibody is a recombinant fusionprotein as meant herein. It may comprise a non-antibody protein and/oran antibody. It may be produced intracellularly or be secreted into theculture medium from which it can be recovered.

The media and methods of the invention can be used to produce just aboutany protein, and is particularly advantageous for proteins whoseexpression is under the control of a strong promoter, such as forexample, a viral promoter, and/or proteins that are encoded on a messagethat has an adenoviral tripartite leader element. Examples of usefulexpression vectors that can be used to produce proteins are disclosed inInternational Application WO 01/27299 and in McMahan et al., (1991),EMBO J. 10: 2821, which describes the pDC409 vector. A protein isgenerally understood to be a protein of at least about 10 amino acids,optionally about 25, 75, or 100 amino acids.

The media and methods of the invention are useful for producingrecombinant proteins. Some proteins that can be produced with themethods of the invention include proteins comprising amino acidsequences identical to or substantially similar to all or part of one ofthe following proteins: a flt3 ligand (as described in InternationalApplication WO 94/28391, incorporated herein by reference), a CD40ligand (as described in U.S. Pat. No. 6,087,329 incorporated herein byreference), erythropoietin, thrombopoeitin, calcitonin, leptin, IL-2,angiopoietin-2 (as described by Maisonpierre et al. (1997), Science277(5322): 55-60, incorporated herein by reference), Fas ligand, ligandfor receptor activator of NF-kappa B (RANKL, as described inInternational Application WO 01/36637, incorporated herein byreference), tumor necrosis factor (TNF)-related apoptosis-inducingligand (TRAIL, as described in International Application WO 97/01633,incorporated herein by reference), thymic stroma-derived lymphopoietin,granulocyte colony stimulating factor, granulocyte-macrophage colonystimulating factor (GM-CSF, as described in Australian Patent No.588819, incorporated herein by reference), mast cell growth factor, stemcell growth factor (described in e.g. U.S. Pat. No. 6,204,363,incorporated herein by reference), epidermal growth factor, keratinocytegrowth factor, megakaryote growth and development factor, RANTES, humanfibrinogen-like 2 protein (FGL2; NCBI accession no. NM_(—)00682; Rüeggand Pytela (1995), Gene 160: 257-62) growth hormone, insulin,insulinotropin, insulin-like growth factors, parathyroid hormone,interferons including α interferons, γ interferon, and consensusinterferons (such as those described in U.S. Pat. Nos. 4,695,623 and4,897,471, both of which are incorporated herein by reference), nervegrowth factor, brain-derived neurotrophic factor, synaptotagmin-likeproteins (SLP 1-5), neurotrophin-3, glucagon, interleukins 1 through 18,colony stimulating factors, lymphotoxin-β, tumor necrosis factor (TNF),leukemia inhibitory factor, oncostatin-M, and various ligands for cellsurface molecules ELK and Hek (such as the ligands for eph-relatedkinases or LERKS). Descriptions of proteins that can be producedaccording to the inventive methods may be found in, for example, HumanCytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwaland Gutterman, eds. Blackwell Sciences, Cambridge, Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook (A. W. Thompson,ed., Academic Press, San Diego, Calif., 1991), all of which areincorporated herein by reference.

Other proteins that can be produced using the media and methods of theinvention include proteins comprising all or part of the amino acidsequence of a receptor for any of the above-mentioned proteins, anantagonist to such a receptor or any of the above-mentioned proteins,and/or proteins substantially similar to such receptors or antagonists.These receptors and antagonists include: both forms of tumor necrosisfactor receptor (TNFR, referred to as p55 and p75, as described in U.S.Pat. No. 5,395,760 and U.S. Pat. No. 5,610,279, both of which areincorporated herein by reference), Interleukin-1 (IL-1) receptors (typesI and II; described in EP Patent No. 0 460 846, U.S. Pat. No. 4,968,607,and U.S. Pat. No. 5,767,064, all of which are incorporated herein byreference), IL-1 receptor antagonists (such as those described in U.S.Pat. No. 6,337,072, incorporated herein by reference), IL-1 antagonistsor inhibitors (such as those described in U.S. Pat. Nos. 5,981,713,6,096,728, and 5,075,222, all of which are incorporated herein byreference) IL-2 receptors, IL-4 receptors (as described in EP Patent No.0 367 566 and U.S. Pat. No. 5,856,296, both of which are incorporated byreference), IL-15 receptors, IL-17 receptors, IL-18 receptors, Fcreceptors, granulocyte-macrophage colony stimulating factor receptor,granulocyte colony stimulating factor receptor, receptors foroncostatin-M and leukemia inhibitory factor, receptor activator ofNF-kappa B (RANK, described in WO 01/36637 and U.S. Pat. No. 6,271,349,both of which are incorporated by reference), osteoprotegerin (describedin e.g. U.S. Pat. No. 6,015,938, incorporated by reference), receptorsfor TRAIL (including TRAIL receptors 1, 2, 3, and 4), and receptors thatcomprise death domains, such as Fas or Apoptosis-Inducing Receptor(AIR).

Other proteins that can be produced using the media and methods of theinvention include proteins comprising all or part of the amino acidsequences of differentiation antigens (referred to as CD proteins) ortheir ligands or proteins substantially similar to either of these. Suchantigens are disclosed in Leukocyte Typing VI (Proceedings of the VIthInternational Workshop and Conference, Kishimoto, Kikutani et al., eds.,Kobe, Japan, 1996, which is incorporated by reference). Similar CDproteins are disclosed in subsequent workshops. Examples of suchantigens include CD22, CD27, CD30, CD39, CD40, and ligands thereto (CD27ligand, CD30 ligand, etc.). Several of the CD antigens are members ofthe TNF receptor family, which also includes 41BB and OX40. The ligandsare often members of the TNF family, as are 41BB ligand and OX40 ligand.Accordingly, members of the TNF and TNFR families can also be purifiedusing the present invention.

Enzymatically active proteins or their ligands can also be producedusing the media and methods of the invention. Examples include proteinscomprising all or part of one of the following proteins or their ligandsor a protein substantially similar to one of these:metalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The media and methods of the invention can also be used to produceantibodies, including human antibodies, or portions thereof and chimericor humanized antibodies. Chimeric antibodies having human constantantibody immunoglobulin domains coupled to one or more murine variableantibody immunoglobulin domain, fragments thereof, or substantiallysimilar proteins. Humanized antibodies contain variable regionscomprising framework portions of human origin and CDR portion from anon-human source. The method of the invention may also be used toproduce conjugates comprising an antibody and a cytotoxic or luminescentsubstance. Such substances include: maytansine derivatives (such asDM1); enterotoxins (such as a Staphlyococcal enterotoxin); iodineisotopes (such as iodine-125); technium isotopes (such as Tc-99m);cyanine fluorochromes (such as Cy5.5.18); and ribosome-inactivatingproteins (such as bouganin, gelonin, or saporin-S6). The invention canalso be used to produce chimeric proteins selected in vitro to bind to aspecific target protein and modify its activity such as those describedin International Applications WO 01/83525 and WO 00/24782, both of whichare incorporated by reference. Examples of antibodies, in vitro-selectedchimeric proteins, or antibody/cytotoxin or antibody/luminophoreconjugates that can be produced by the methods of the invention includethose that recognize any one or a combination of proteins including, butnot limited to, the above-mentioned proteins and/or the followingantigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25,CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1α, IL-1β,IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor,IL-6 receptor, IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-β andanalogs thereof (such as those described in U.S. Pat. Nos. 5,272,064 and5,149,792), VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (including thosedescribed in U.S. Pat. No. 6,235,883 B1, incorporated by reference) VEGFreceptor, hepatocyte growth factor, osteoprotegerin ligand, interferongamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1,and zTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev.13(1): 19-25), C5 complement, IgE, tumor antigen CA125, tumor antigenMUC1, PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, a tumor-associated glycoproteinTAG-72, the SK-1 antigen, tumor-associated epitopes that are present inelevated levels in the sera of patients with colon and/or pancreaticcancer, cancer-associated epitopes or proteins expressed on breast,colon, squamous cell, prostate, pancreatic, lung, and/or kidney cancercells and/or on melanoma, glioma, or neuroblastoma cells, the necroticcore of a tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, theadhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, L-selectin, Respiratory Syncitial Virus, human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), Streptococcus mutans, andStaphlycoccus aureus.

The media and methods of the invention may also be used to produce allor part of an anti-idiotypic antibody or a substantially similarprotein, including anti-idiotypic antibodies against: an antibodytargeted to the tumor antigen gp72; an antibody against the gangliosideGD3; an antibody against the ganglioside GD2; or antibodiessubstantially similar to these.

The media and methods of the invention can also be used to producerecombinant fusion proteins comprising any of the above-mentionedproteins. For example, recombinant fusion proteins comprising one of theabove-mentioned proteins plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an antibody, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Håkansson et al. (1999), Structure7:255-64, all of which are incorporated by reference. Specificallyincluded among such recombinant fusion proteins are proteins in which aportion of TNFR or RANK is fused to an Fc portion of an antibody(TNFR:Fc or RANK:Fc). TNFR:Fc comprises the Fc portion of an antibodyfused to an extracellular domain of TNFR, which includes amino acidsequences substantially similar to amino acids 1-163, 1-185, or 1-235 ofFIG. 2A of U.S. Pat. No. 5,395,760, which is incorporated by reference.RANK:Fc is described in International Application WO 01/36637, which isincorporated by reference.

Preferably, the proteins are expressed under the control of aheterologous control element such as, for example, a promoter that doesnot in nature direct the production of that protein. For example, thepromoter can be a strong viral promoter (e.g., CMV, SV40) that directsthe expression of a mammalian protein. The host cell may or may notnormally produce the protein. For example, the host cell can be a CHOcell that has been genetically engineered to produce a human protein,meaning that nucleic acid encoding the human protein has been introducedinto the CHO cell. Alternatively, the host cell can be a human cell thathas been genetically engineered to produce increased levels of a humanprotein normally present only at very low levels (e.g., by replacing theendogenous promoter with a strong viral promoter). For the production ofrecombinant proteins, an expression vector encoding the recombinantprotein can be transferred, for example by transfection or viralinfection, into a substantially homogeneous culture of host cells. Theexpression vector, which can be constructed using the methods of geneticengineering, can include nucleic acids encoding the protein of interestoperably linked to suitable regulatory sequences.

The regulatory sequences are typically derived from mammalian,microbial, viral, and/or insect genes. Examples of regulatory sequencesinclude transcriptional promoters, operators, and enhancers, a ribosomalbinding site (see e.g. Kozak (1991), J. Biol. Chem. 266:19867-19870),appropriate sequences to control transcriptional and translationalinitiation and termination, polyadenylation signals (see e.g. McLauchlanet al. (1988), Nucleic Acids Res. 16:5323-33), and matrix and scaffoldattachment sites (see Phi-Van et al. (1988), Mol. Cell. Biol.10:2302-07; Stief et al. (1989), Nature 341:342-35; Bonifer et al.(1990), EMBO J. 9:2843-38). Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the protein codingsequence. Thus, a promoter nucleotide sequence is operably linked to aprotein coding sequence if the promoter nucleotide sequence controls thetranscription of the coding sequence. A gene encoding a selectablemarker is generally incorporated into the expression vector tofacilitate the identification of recombinant cells.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter and enhancer sequences are derived from polyoma virus,adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV).For example, the human CMV promoter/enhancer of immediate early gene 1may be used. See e.g. Patterson et al. (1994), Applied Microbiol.Biotechnol. 40:691-98. DNA sequences derived from the SV40 viral genome,for example, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites can be used to provide other genetic elements forexpression of a structural gene sequence in a mammalian host cell. Viralearly and late promoters are particularly useful because both are easilyobtained from a viral genome as a fragment, which can also contain aviral origin of replication (Fiers et al. (1978), Nature 273:113;Kaufman (1990), Meth. in Enzymol. 185:487-511). Smaller or larger SV40fragments can also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

In addition, a sequence encoding an appropriate native or heterologoussignal peptide (leader sequence) can be incorporated into the expressionvector, to promote extracellular secretion of the recombinant protein.The signal peptide will be cleaved from the recombinant protein uponsecretion from the cell. The choice of signal peptide or leader dependson the type of host cells in which the recombinant protein is to beproduced. Examples of signal peptides that are functional in mammalianhost cells include the signal sequence for interleukin-7 (IL-7)described in U.S. Pat. No. 4,965,195, the signal sequence forinterleukin-2 receptor described in Cosman et al. (1984), Nature312:768; the interleukin-4 receptor signal peptide described in EPPatent No. 367,566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; and the type II interleukin-1receptor signal peptide described in EP Patent No. 0 460 846.

Established methods for introducing DNA into mammalian cells have beendescribed. Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69. Additional protocols using commercially available reagents, suchas the cationic lipid reagents LIPOFECTAMINE™, LIPOFECTAMINE™-2000, orLIPOFECTAMINE™-PLUS (which can be purchased from Invitrogen), can beused to transfect cells. Felgner et al. (1987)., Proc. Natl. Acad. Sci.USA 84:7413-7417. In addition, electroporation or bombardment withmicroprojectiles coated with nucleic acids can be used to transfectmammalian cells using procedures, such as those in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1-3, Cold SpringHarbor Laboratory Press (1989) and Fitzpatrick-McElligott (1992),Biotechnology (NY) 10(9):1036-40. Selection of stable transfectants canbe performed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al. ((1990), Meth. inEnzymology 185:487-511), describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR.Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA 77:4216-4220. Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Pat.Nos. 6,312,951 B1, 6,027,915, and 6,309,841 B1) and the tripartiteleader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al. (1982),J. Biol. Chem. 257:13475-13491). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow (1993), Current Opinion in Genetics andDevelopment 3:295-300; Ramesh et al. (1996), Nucleic Acids Research24:2697-2700). Expression of a heterologous cDNA as part of adicistronic mRNA followed by the gene for a selectable marker (e.g.DHFR) has been shown to improve transfectability of the host andexpression of the heterologous cDNA (Kaufman et al. (1990), Methods inEnzymol. 185:487-511). Exemplary expression vectors that employdicistronic mRNAs are pTR-DC/GFP described by Mosser et al.,Biotechniques 22:150-161 (1997), and p2A51 described by Morris et al.,in Animal Cell Technology, pp. 529-534 (1997).

A useful high expression vector, pCAVNOT, has been described by Mosleyet al. ((1989), Cell 59:335-348). Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg((1983), Mol. Cell. Biol. 3:280). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. ((1986),Mol. Immunol. 23:935). A useful high expression vector, PMLSV N1/N4,described by Cosman et al. ((1984), Nature 312:768), has been depositedas ATCC 39890. Additional useful mammalian expression vectors aredescribed in EP Patent No.-A-0 367 566 and WO 01/27299 A1. The vectorscan be derived from retroviruses. Further, other vectors, such as thosedescribed by Aldrich et al. (Biotechnol. Prog. 19: 1433-38 (2003)) or byBianchi and McGrew (Biotechnol. Bioeng. 84: 439-44 (2003)), may be used.

In place of the native signal sequence, a heterologous signal sequencecan be added, such as one of the following sequences: the signalsequence for IL-7 described in U.S. Pat. No. 4,965,195; the signalsequence for IL-2 receptor described in Cosman et al. (Nature 312:768(1984)); the IL-4 signal peptide described in EP Patent No. 0 367 566;the type I IL-1 receptor signal peptide described in U.S. Pat. No.4,968,607; and the type II IL-1 receptor signal peptide described in EPPatent No. 0 460 846.

The proteins can be produced recombinantly in eukaryotic cells and canbe secreted by host cells adapted to grow in cell culture. Optionally,host cells for use in the invention are mammalian cells. The cells canbe also genetically engineered to express a gene of interest, can bemammalian production cells adapted to grow in cell culture, and/or canbe homogenous cell lines. Examples of such cells commonly used in theindustry are VERO, BHK, HeLa, CV1 (including Cos), MDCK, 293, 3T3,myeloma cell lines (e.g., NSO, NS1), PC12, WI38 cells, and Chinesehamster ovary (CHO) cells, which are widely used for the production ofseveral complex recombinant proteins, e.g. cytokines, clotting factors,and antibodies (Brasel et al. (1996), Blood 88:2004-2012; Kaufman et al.(1988), J. Biol Chem 263:6352-6362; McKinnon et al. (1991), J MolEndocrinol 6:231-239; Wood et al. (1990), J. Immunol. 145:3011-3016).The dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaubet al. (1980), Proc Natl Acad Sci USA 77: 4216-4220, which isincorporated by reference), DXB 11 and DG-44, are desirable CHO hostcell lines because the efficient DHFR selectable and amplifiable geneexpression system allows high level recombinant protein expression inthese cells (Kaufman R. J. (1990), Meth Enzymol 185:537-566, which isincorporated by reference). In addition, these cells are easy tomanipulate as adherent or suspension cultures and exhibit relativelygood genetic stability. CHO cells and recombinant proteins expressed inthem have been extensively characterized and have been approved for usein clinical commercial manufacturing by regulatory agencies. The methodsof the invention can also be practiced using hybridoma cell lines thatproduce an antibody. Methods for making hybridoma lines are well knownin the art. See e.g. Berzofsky et al. in Paul, ed., FundamentalImmunology, Second Edition, pp. 315-356, at 347-350, Raven Press Ltd.,New York (1989). Cell lines derived from the above-mentioned lines arealso suitable for practicing the invention.

According to the present invention, a eukaryotic, optionally amammalian, host cell is cultured under conditions that promote theproduction of the protein of interest, which can be any protein,including an antibody or a recombinant protein. The culture is fed usingthe concentrated feed media and methods of the invention.

Cell culture medium formulations for use as base media are well known inthe art. To these basal culture medium formulations the skilled artisanwill add components such as amino acids, salts, sugars, vitamins,hormones, growth factors, buffers, antibiotics, lipids, trace elementsand the like, depending on the requirements of the host cells to becultured. The culture medium may or may not contain serum and/orprotein. Various tissue culture media, including serum-free and/ordefined culture media, are commercially available for cell culture.Tissue culture media is defined, for purposes of the invention, as amedia suitable for growth of eukaryotic cells, and optionally mammaliancells, in in vitro cell culture. Typically, tissue culture mediacontains a buffer, salts, energy source, amino acids, vitamins and traceessential elements. Any media capable of supporting growth of theappropriate eukaryotic cell in culture can be used; the invention isbroadly applicable to eukaryotic cells in culture, particularlymammalian cells, and the choice of media is not crucial to theinvention. Tissue culture media suitable for use as a base medium, asdefined herein, in the methods of the invention are commerciallyavailable from, e.g., ATCC (Manassas, Va.). For example, any one orcombination of the following media can be used as a base medium:RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium(DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium,Iscove's Modified Dulbecco's Medium, McCoy's 5A Medium, Leibovitz's L-15Medium, and serum-free media such as EX-CELL™ 300 Series (available fromSAFC Biosciences, which was formerly JRH Biosciences, Lenexa, Kans.,USA), among others, which can be obtained from the American Type CultureCollection or SAFC Biosciences, as well as other vendors. When definedmedium that is serum-free and/or peptone-free is used, the medium isusually highly enriched for amino acids and trace elements. See, forexample, U.S. Pat. Nos. 5,122,469 to Mather et al. and 5,633,162 to Keenet al. Peptone or other protein hydrolysates can be added to a culturemedium.

Cell culture media, including base media and/or feed media, can beserum-free, protein-free, growth factor-free, and/or peptone-free media.The term “serum-free” as applied to media includes any mammalian cellculture medium that does not contain serum, such as fetal bovine serum.The term “insulin-free” as applied to media includes any medium to whichno exogenous insulin has been added. By exogenous is meant, in thiscontext, other than that produced by the culturing of the cellsthemselves. The term “IGF-1-free” as applied to media includes anymedium to which no exogenous Insulin-like growth factor-1 (IGF-1) oranalog (such as, for example, LongR3, [Ala31], or [Leu24] [Ala31] IGF-1analogs available from Novozymes GroPep Ltd. of Thebarton, SouthAustralia) has been added. The term “growth-factor free” as applied tomedia includes any medium to which no exogenous growth factor (e.g.,insulin, IGF-1) has been added. The term “protein-free” as applied tomedia includes medium free from exogenously added protein, such as, forexample, transferrin and the protein growth factors IGF-1 and insulin.Protein-free media may or may not have peptones. The term “peptone-free”as applied to media includes any medium to which no exogenous proteinhydrolysates have been added such as, for example, animal and/or plantprotein hydrolysates. Eliminating peptone from media has the advantagesof reducing lot to lot variability and enhancing processing steps, suchas filtration. Chemically defined media are media in which everycomponent is defined and obtained from a pure source, preferably anon-animal source.

The skilled artisan may also choose to use one of the manyindividualized base media formulations that have been developed tomaximize cell growth, cell viability, and/or recombinant proteinproduction in a particular cultured host cell. The methods according tothe current invention may be used in combination with commerciallyavailable cell culture media or with a cell culture medium that has beenindividually formulated for use with a particular cell line. Forexample, an enriched base medium that could support increased proteinproduction may comprise a mixture of two or more commercial media, suchas, for instance, DMEM and Ham's F12 media combined in ratios such as,for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or even up to 1:15or higher. Alternatively or in addition, a base medium can be enrichedby the addition of nutrients, such as amino acids or peptone, and/or amedium (or most of its components with the exceptions noted below) canbe used at greater than its usual, recommended concentration, forexample at about 2×, 3×, 4×, 5×, 6×, 7×, 8×, or even higherconcentrations. As used herein, “1×” means the standard concentrationnormally used in a particular base medium. “2×” means twice the standardconcentration, etc. In any of these embodiments, medium components thatcan substantially affect osmolarity, such as salts, cannot be increasedin concentration so that the osmolarity of the culture falls outside ofan acceptable range, such as, for example about 200-700 mOsm, or, moretypically, about 270 mOsm to about 400 mOsm. Thus, a base medium may,for example, be 8× with respect to all components except salts, whichcan be present at only 1×. An enriched medium may be serum free and/orprotein free. A serum free medium lacks serum, such as, for example,bovine serum, which is commonly used in cell culture. Further, a basemedium may be supplemented periodically with a feed medium of theinvention during the time a culture is maintained to replenish mediumcomponents that can become depleted such as, for example, vitamins,amino acids, and metabolic precursors. As is known in the art, differentmedia and temperatures may have somewhat different effects on differentcell lines, and the same medium and temperature may not be suitable forall cell lines.

Concentrated feed media of the invention can be based on just about anybase culture medium. Such a concentrated feed medium can contain many ofthe components of the culture medium at, for example, about 5×, 6×, 7×,8×, 9×, 10×, 12×, 14×, 16×, 20×, 30×, 50×, 100×, 200×, 400×, 600×, 800×,or even about 1000× of their normal amount. However, not all componentsof a standard medium can be increased in concentration in a concentratedfeed medium to the same extent, if at all. For example, in a 10×concentrated feed medium, salts such as sodium chloride or iron saltsmight be present at only 1× or be entirely absent. Using them at 10×concentration could bring the osmolarity of the medium into anunacceptable range. An acceptable osmotic range can be from about200-1500 mOsm. Similarly, some medium components are not substantiallydepleted during cell growth and, thus, need not be present at increasedconcentrations, or, in some cases need not be present at all, in a feedmedium. Guided by these considerations, one of skill in the art couldreadily design a concentrated feed medium based on any known eukaryotic,especially mammalian, base culture medium.

As is known in the art the solubility and stability of a concentratedfeed medium can be very substantially affected by relatively insolublemedium components, such as tyrosine and cystine, or by medium componentsthat readily convert to insoluble species, such as cysteine. As shown inExample 1, tyrosine and cysteine can be important for increasing proteinproduction by a culture. Thus, it can be desirable to include suchinsoluble species at high concentrations in a feed medium. The media andmethods of the invention provide a means, i.e., the addition of highconcentrations of sodium pyruvate, of stabilizing media containing highconcentrations of cysteine and/or cystine and/or tyrosine.

Suitable culture conditions for mammalian cells are known in the art.See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed.,Oxford university press, New York (1992). Mammalian cells may becultured in suspension or while attached to a solid substrate.Furthermore, mammalian cells may be cultured, for example, in fluidizedbed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks,or stirred tank bioreactors, with or without microcarriers, and operatedin a batch, fed batch, continuous, semi-continuous, or perfusion mode.The media and methods of the invention specifically involve a method inwhich concentrated feed medium is added to the culture eithercontinuously or at intervals during the culture. For example, a culturemay be fed, for example, once per day, every other day, every threedays, every four days, every five days, or may be fed when theconcentration of a specific medium component, which is being monitored,falls outside a desired range. Alternatively, a culture may be fed on anirregular schedule, for example on days 2, 5, and 7.

The methods according to the present invention may be used to improvethe production of recombinant proteins in both single phase and multiplephase culture processes. In a single phase process, cells are inoculatedinto a culture environment and the disclosed methods are employed duringthe single production phase. In a multiple stage process, cells arecultured in two or more distinct phases. For example cells may becultured first in one or more growth phases, under environmentalconditions that maximize cell proliferation and viability, thentransferred to a production phase, under conditions that maximizeprotein production. In a commercial process for production of a proteinby mammalian cells, there are commonly multiple, for example, at leastabout 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases that occur indifferent culture vessels preceding a final production phase. The growthand production phases may be preceded by, or separated by, one or moretransition phases. In multiple phase processes, the methods according tothe present invention can be employed at least during the productionphase, although they may also be employed in a preceding growth phase. Aproduction phase can be conducted at large scale. A large scale processcan be conducted in a volume of at least about 100, 500, 1000, 2000,3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters, or larger. Agrowth phase may occur at a higher temperature than a production phase.For example, a growth phase may occur at a first temperature from about35° C. to about 39° C., and a production phase may occur at a secondtemperature from about 29° C. to about 39° C., optionally from about 30°C. to about 36° C. or from about 30° C. to about 34° C. Chemicalinducers of protein production, such as, for example, caffeine,butyrate, and hexamethylene bisacetamide (HMBA), may be added at thesame time as, before, and/or after a temperature shift. If inducers areadded after a temperature shift, they can be added from one hour to fivedays after the temperature shift, optionally from one to two days afterthe temperature shift.

The methods of the invention can be used to culture cells that produce aprotein, and the resulting expressed protein can then be collected. Inaddition, the protein can be purified, or partially purified, from sucha culture (e.g., from culture medium or cell extracts) using knownprocesses. By “partially purified” means that some fractionationprocedure, or procedures, have been carried out, but that more proteinspecies (at least 10%) than the desired protein is present. By“purified” is meant that the protein is essentially homogeneous, i.e.,less than about 2% contaminating proteins are present. Fractionationprocedures can include but are not limited to one or more steps offiltration, centrifugation, precipitation, phase separation, affinitypurification, gel filtration, ion exchange chromatography, hydrophobicinteraction chromatography (HIC; using such resins as phenyl ether,butyl ether, or propyl ether), HPLC, or some combination of above.

For example, the purification of the protein can include an affinitycolumn containing agents which will bind to the protein; one or morecolumn steps over such affinity resins as concanavalin A-agarose,heparin-TOYOPEARL® (Toyo Soda Manufacturing Co., Ltd., Japan) orCibacrom blue 3GA SEPHAROSE®. (Pharmacia Fine Chemicals, Inc., NewYork); one or more steps involving elution; and/or immunoaffinitychromatography. The protein can be expressed in a form that facilitatespurification. For example, it may be expressed as a fusion protein, suchas those of maltose binding protein (MBP), glutathione-S-transferase(GST), or thioredoxin (TRX). Kits for expression and purification ofsuch fusion proteins are commercially available from New England BioLab(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen,respectively. The protein can be tagged with an epitope and subsequentlypurified by using a specific antibody directed to such epitope. One suchepitope (FLAG®) is commercially available from Kodak (New Haven, Conn.).It is also possible to utilize an affinity column comprising aprotein-binding protein, such as a monoclonal antibody to therecombinant protein, to affinity-purify expressed proteins. Other typesof affinity purification steps can be a Protein A or a Protein G column,which affinity agents bind to proteins that contain Fc domains. Proteinscan be removed from an affinity column using conventional techniques,e.g., in a high salt elution buffer and then dialyzed into a lower saltbuffer for use or by changing pH or other components depending on theaffinity matrix utilized, or can be competitively removed using thenaturally occurring substrate of the affinity moiety.

The desired degree of final purity depends on the intended use of theprotein. A relatively high degree of purity is desired when the proteinis to be administered in vivo, for example. In such a case, the proteinsare purified such that no protein bands corresponding to other proteinsare detectable upon analysis by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). It will be recognized by one skilled in the pertinent fieldthat multiple bands corresponding to the protein can be visualized bySDS-PAGE, due to differential glycosylation, differentialpost-translational processing, and the like. Optionally, the protein ofthe invention is purified to substantial homogeneity, as indicated by asingle protein band upon analysis by SDS-PAGE. The protein band can bevisualized by silver staining, Coomassie blue staining, or (if theprotein is radiolabeled) by autoradiography.

The invention also optionally encompasses further formulating theproteins. By the term “formulating” is meant that the proteins can bebuffer exchanged, sterilized, bulk-packaged, and/or packaged for a finaluser. For purposes of the invention, the term “sterile bulk form” meansthat a formulation is free, or essentially free, of microbialcontamination (to such an extent as is acceptable for food and/or drugpurposes), and is of defined composition and concentration. The term“sterile unit dose form” means a form that is appropriate for thecustomer and/or patient administration or consumption. Such compositionscan comprise an effective amount of the protein, in combination withother components such as a physiologically acceptable diluent, carrier,or excipient. The term “physiologically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s).

Formulations suitable for administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents or thickening agents.The proteins can be formulated according to known methods used toprepare pharmaceutically useful compositions. They can be combined inadmixture, either as the sole active material or with other known activematerials suitable for a given indication, with pharmaceuticallyacceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphatebuffered solutions), preservatives (e.g., thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants, and/or carriers.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Company, Easton, Pa. In addition, such compositions can becomplexed with polyethylene glycol (PEG), metal ions, or incorporatedinto polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. Nos.4,235,871, 4,501,728, 4,837,028, and 4,737,323. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance, and are thus chosen according tothe intended application, so that the characteristics of the carrierwill depend on the selected route of administration. Sustained-releaseforms suitable for use include, but are not limited to, proteins thatare encapsulated in a slowly-dissolving biocompatible polymer (such asthe alginate microparticles described in U.S. Pat. No. 6,036,978),admixed with such a polymer (including topically applied hydrogels), andor encased in a biocompatible semi-permeable implant.

All references cited herein are incorporated by reference herein intheir entirety. The invention having been described, the followingexamples are offered by way of illustration, and not limitation.

EXAMPLES Example 1 Addition of Tyrosine and Cysteine Increases ProteinTiters

A CHO cell line producing a recombinant protein was cultured in acommercially available base medium (a modified version of the EX-CELL™325 PF CHO serum free medium of catalog number 24340C from SAFCBiosciences (Lenexa, Kans.) in which the sodium chloride concentrationis 4.5 g/L, rather than the 6.508 g/L present in catalog number 24340C)and fed with Feed Medium A with or without cysteine and/or tyrosine.Feed Medium A was designed based on the standard, commercially availablemedium, DMEM. DMEM is commercially available from, for example, AmericanType Culture Collection, Manassas, Va., USA. Feed Medium A contained allof the ingredients listed in Table 1 within the concentration rangeslisted in Table 1 except the following ingredients, which were not partof Feed Medium A: arginine, asparagine, biotin, calcium chloride, cupricsulfate, cyanocobalamin, cysteine, cystine, glucose (which was addedseparately), hypoxanthine, linoleic acid, alpha-lipoic acid, magnesiumchloride, niacinamide, potassium chloride, potassium phosphatemonobasic, potassium phosphate disbasic, proline, putrescine, pyridoxal,riboflavin, sodium phosphate dibasic, sodium pyruvate, thymidine,tyrosine, and zinc sulfate. The amount of sodium chloride in Feed MediumA was adjusted so as to bring the osmolarity of the medium to about 300mOsm.

To Feed Medium A, cysteine, tyrosine, and sodium pyruvate were added invarying combinations. The feed media differed only in theirconcentrations of tyrosine, cysteine, and sodium pyruvate, which areshown in Table 2 below. The feed media were mixed at room temperature.The cells were cultured in 125 ml shaker flasks with vented caps in thebase medium described above. The shakers flasks were maintained at 36°C. in 5% CO₂. Flasks were agitated at 150 rpm. All flasks were seeded5×10⁵ cells per ml in a volume of 30 ml volume. On days 3, 6, and 8, 0.1culture volume of each the feed media described in Table 2 were added toone of the four flasks. Day 0 is the day the cultures were started, day1 is the first day thereafter, day 2 is the second day thereafter, etc.Protein titer was determined at days 8 and 10.

TABLE 2 Concentrations of Medium Components Feed Tyrosine CysteineSodium pyruvate Medium (mM) (mM) (mM) #1 0.0 0.0 0.0 #2 0.0 0.0 36.35 #34.40 12.53 0.0 #4 4.40 12.53 36.35

FIG. 1 shows the protein titers observed on days 8 and 10 of theculture. These data indicate that the addition of tyrosine and cysteineto Feed Medium A increases protein titer. Thus, adding cysteine andtyrosine to a feed medium can be advantageous. Hence, methods forkeeping these medium components in solution can be advantageous,particularly for large volumes of feed media and/or in situations whereit is most convenient to make feed media somewhat in advance of itsactual use.

Example 2 High Concentrations of Pyruvate Stabilize Concentrated FeedMedia Containing Tyrosine, Cysteine, and Cystine

Feed Medium B is a concentrated feed medium. It contains all of theingredients listed in Table 1 within the concentration ranges listed inTable 1 except the following ingredients, which were not part of FeedMedium B: arginine, glucose (which is added to the culture separately),and ferric nitrate. Feed Medium B contains 1 mM cystine, 3.03 mMtyrosine, 3.5 mM sodium pyruvate, and 7 mM cysteine. Feed Medium Brequired heating to 45° C. in order to dissolve all medium components,and it typically formed a precipitate upon storage for a few days atroom temperature or at 4-8° C. This characteristic made its use,especially for a large-scale commercial culture for production of abiologic, certainly very inconvenient, if not potentially impractical.

To circumvent the issues of insolubility and instability, tyrosine andcystine were eliminated from the Feed Medium B. The resulting medium,Feed Medium C, did not require heating in order to dissolve and did notprecipitate within 28 days of storage at 4° C. Thus, tyrosine and/orcystine are likely responsible for the insolubility and instability ofFeed Medium B.

Varying concentrations of sodium pyruvate were added to Feed Medium B todetermine whether Feed Medium B could be stabilized by pyruvate. Themedia were stored at 4-8° C. and were checked for stability on days 3,6, and 8, where day 0 is the day the medium is made and day 1 is thenext day. The volume of each sample was 50 ml. Table 3 below shows theconcentrations of cysteine, cystine, tyrosine, and pyruvate in eachtested medium.

TABLE 3 Concentrations of medium components and stability of mediaCysteine Cystine Tyrosine Pyruvate (mM) (mM) (mM) (mM) 7.00 1.0 3.033.50 7.00 1.0 3.03 11.36 7.00 1.0 3.03 14.00 7.00 1.0 3.03 19.27 7.001.0 3.03 35.00On day 3, the sample containing 3.5 mM pyruvate contained moreprecipitate than any other, followed by the sample containing 11.36 mMpyruvate and by the sample containing 14 mM pyruvate. Samples containing19.27 and 35 mM pyruvate did not contain precipitate after one week ofstorage. However, the sample containing 19.27 mM pyruvate did contain aprecipitate when it was discarded some weeks later. The samplecontaining 35 mM pyruvate was still without precipitate at this time.These data indicate that a concentration of 19.27 mM is adequate tostabilize Feed Medium B for at least one week and that a concentrationof 35 mM pyruvate can stabilize Feed Medium B somewhat longer that oneweek.

Example 3 Stabilizing a Feed Medium Containing Tyrosine and Cysteine

In order to produce a stable medium containing tyrosine, varyingconcentrations of pyruvate and tyrosine were added to Feed Medium C.These concentrations are shown in Table 4 below. Feed Medium C, containsall the ingredients listed in Table 1 within the concentration rangeslisted in Table 1 except the following ingredients, which were not partof Feed Medium C: arginine, cystine, glucose (which is added to theculture separately), ferric nitrate, and tyrosine. Thus, as mentionedabove, the only difference between Feed Media B and C is the absence ofcystine and tyrosine in Feed Medium C. The concentration of cysteine inFeed Medium C is 7 mM.

Stability during storage was tested. The media were stored at 4-8° C.The media were evaluated for precipitation by visual inspection on days0, 1, 2, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, and 28,where day 0 is the day the media was made, day 1 is the first daythereafter, day 2 is the second day thereafter, etc. Media samplesscored “none” in the rightmost column of Table 4 did not contain anyprecipitate while this experiment was ongoing, that is, for 28 days. Forsamples that did precipitate during the course of the experiment, thefirst day on which a precipitate was observed is recorded in therightmost column of Table 4.

TABLE 4 Stabilizing effects of sodium pyruvate at varying concentrationsof tyrosine Tyrosine Sodium pyruvate Day on which concentrationconcentration precipitate was Media (mM) (mM) first noted Feed Medium C0 3.5 none Feed Medium C 3.03 3.5 none Feed Medium C 3.03 35.0 none FeedMedium C 4.40 3.5 19  Feed Medium C 4.40 35.0 none Feed Medium C 5.743.5 9 Feed Medium C 5.74 35.0 6

These data indicate that 35 mM sodium pyruvate stabilizes Feed Medium Cwhen it contains 4.4 mM tyrosine, whereas the lower pyruvateconcentration, 3.5 mM, does not. However, addition of 3.03 mM tyrosinedid not destabilize the medium, and even feed medium containing 4.4 mMtyrosine was stable for a considerable period when it contained 3.5 mMpyruvate. No concentration of pyruvate tested stabilized mediumcontaining 5.74 mM tyrosine.

Example 4 Effects of Cysteine, Cystine, Tyrosine, Pyruvate, and StorageTemperature on Medium Stability

The following experiment explores interactions of various parameters andtheir effects on the stability of Feed Medium C. Feed Medium C (which isdescribed in Examples 2 and 3) was prepared, and tyrosine, sodiumpyruvate, cysteine, and cystine were added, or not, to various mediasamples. All media were mixed at room temperature. Stability wasassessed at either room temperature or 4-8° C. The samples were visuallyinspected on days 0, 2, 5, 7, 9, 12, 14, 16, 19, 20, 23, and 26 ofstorage to determine whether precipitates had formed. Table 5 belowlists the storage temperatures (where “RT” means room temperature) andthe concentrations of cysteine, cystine, tyrosine, and sodium pyruvatein each sample. Samples are scored for precipitate as explained inExample 3, except that “none” in the rightmost column means that therewas no precipitate for the 26 days (rather than the 28 days) that theexperiment was ongoing.

TABLE 5 Stability and composition of media Sodium Day on whichL-Cysteine L-Tyrosine Pyruvate L-Cystine Storage Osmo precipitate was(mM) (mM) (mM) (mM) Temperature pH (mOsm) first noted 7.0 0.0 3.5 0.0 RT6.52 238 none 7.0 0.0 35.0 1.0 RT 6.19 294 none 7.0 4.4 3.5 1.0 RT 7.06252 2 7.0 4.4 35.0 0.0 RT 6.87 307 none 16 0.0 3.5 1.0 RT 6.01 263 2 160.0 35.0 0.0 RT 6.01 303 none 16 4.4 3.5 0.0 RT 6.48 271 2 16 4.4 35.01.0 RT 6.01 308 none 7.0 0.0 3.5 1.0 RT 6.52 238 2 7.0 0.0 35.0 0.0 RT6.31 283 none 7.0 4.4 3.5 0.0 RT 7.11 250 none 7.0 4.4 35.0 1.0 RT 6.83289 none 16 0.0 3.5 0.0 RT 6.26 256 2 16 0.0 35.0 1.0 RT 6.01 299 none16 4.4 3.5 1.0 RT 6.39 262 2 16 4.4 35.0 0.0 RT 6.01 317 none 7.0 0.03.5 1.0 4-8° C. 6.51 237 2 7.0 0.0 35.0 0.0 4-8° C. 6.32 284 none 7.04.4 3.5 0.0 4-8° C. 7.07 252 23 7.0 4.4 35.0 1.0 4-8° C. 6.81 294 none16 0.0 3.5 0.0 4-8° C. 6.45 259 2 16 0.0 35.0 1.0 4-8° C. 6.11 300 none16 4.4 3.5 1.0 4-8° C. 6.37 266 2 16 4.4 35.0 0.0 4-8° C. 6.01 316 none7.0 0.0 3.5 0.0 4-8° C. 6.55 242 none 7.0 0.0 35.0 1.0 4-8° C. 6.19 296none 7.0 4.4 3.5 1.0 4-8° C. 7.04 253 2 7.0 4.4 35.0 0.0 4-8° C. 6.84315 none 16 0.0 3.5 1.0 4-8° C. 6.01 260 2 16 0.0 35.0 0.0 4-8° C. 6.01304 none 16 4.4 3.5 0.0 4-8° C. 6.48 269 2 16 4.4 35.0 1.0 4-8° C. 6.01310 none

Data were entered into the software JMP® (SAS Institute Inc., Cary,N.C.), which produced FIGS. 2A and 2B. FIGS. 2A and 2B and Table 4 makea number of trends in the data obvious. First, media were approximatelyequally stable at room temperature or at refrigerator temperature, i.e.,4-8° C. The presence or absence of tyrosine within the tested range,that is, either 0.0 mM or 4.4 mM, had little or no effect on mediumstability. However, higher concentrations of cysteine, cystine, or acombination of the two led to precipitation of media at low (3.5 mM),but not at high (35 mM), concentrations of sodium pyruvate. Thus, theaddition of higher concentrations of sodium pyruvate can stabilize mediacontaining the higher concentrations of cysteine and/or cystine testedin this experiment.

Example 5 Stabilizing Effects of Pyruvate on Feed Medium A

Using the Design of Experiment (DOE) portion of JMP® software (SASInstitute Inc., Cary, N.C.), an experiment was designed to measure thestability of Medium A (described above in Example 1) with varyingamounts of added cystine, cysteine, tyrosine, and sodium pyruvate. Theexperiment tested the effects of (1) 0.0 and 1.12 mM cystine, (2) 0.0,3.83, and 4.59 mM tyrosine, (3) 0.0, 6.83, and 12.53 mM cysteine, (4)4.54, 9.09, 18.18, and 36.35 mM sodium pyruvate, and (5) all possiblecombinations thereof on medium stability. The media was compounded atroom temperature, and its stability was assessed at 4-8° C. at days 0,2, 4, 6, 9, 12, 14, 16, 19, and 21 days. Stability is recorded in Table6, below, as explained in Example 3. Samples marked “none” had noprecipitate on day 21, when the experiment was concluded.

TABLE 6 Stability of Feed Medium A with various additives Day on whichSodium precipitate Cystine Tyrosine Cysteine pyruvate Osmo was (mM) (mM)(mM) (mM) pH (mOsm) first noted 0 0 0 4.54 6.99 299 none 1.12 0 0 4.546.97 287 none 0 3.83 0 4.54 7.01 296 none 1.12 3.83 0 4.54 6.99 300 none0 4.59 0 4.54 6.98 283 2 1.12 4.59 0 4.54 7.00 300 2 0 0 6.83 4.54 6.97300 none 1.12 0 6.83 4.54 6.97 293 4 0 3.83 6.83 4.54 7.01 288 none 1.123.83 6.83 4.54 7.00 294 2 0 4.59 6.83 4.54 6.99 297 2 1.12 4.59 6.834.54 6.99 300 2 0 0 12.53 4.54 6.95 292 6 1.12 0 12.53 4.54 6.94 288 2 03.83 12.53 4.54 7.05 300 4 1.12 3.83 12.53 4.54 6.92 291 2 0 4.59 12.534.54 6.94 305 2 1.12 4.59 12.53 4.54 6.94 302 2 0 0 0 9.09 6.99 299 none1.12 0 0 9.09 6.93 293 none 0 3.83 0 9.09 7.01 309 none 1.12 3.83 0 9.096.99 304 none 0 4.59 0 9.09 6.94 303 4 1.12 4.59 0 9.09 6.99 308 2 0 06.83 9.09 7.02 303 none 1.12 0 6.83 9.09 7.01 293 4 0 3.83 6.83 9.096.89 294 none 1.12 1.12 6.83 9.09 6.98 302 2 0 4.59 6.83 9.09 6.95 302 21.12 4.59 6.83 9.09 7.01 306 2 0 0 12.53 9.09 6.97 300 21  1.12 0 12.539.09 6.99 299 2 0 3.83 12.53 9.09 6.99 303 6 1.12 3.83 12.53 9.09 7.01307 2 0 4.59 12.53 9.09 6.99 310 2 1.12 4.59 12.53 9.09 7.00 311 2 0 0 018.18 6.97 312 none 1.12 0 0 18.18 7.02 310 none 0 3.83 0 18.18 6.99 312none 1.12 3.83 0 18.18 6.98 317 none 0 4.59 0 18.18 7.02 303 2 1.12 4.590 18.18 7.03 316 2 0 0 6.83 18.18 6.97 296 none 1.12 0 6.83 18.18 7.02303 6 0 3.83 6.83 18.18 7.03 316 none 1.12 3.83 6.83 18.18 6.98 310 4 04.59 6.83 18.18 6.98 309 4 1.12 4.59 6.83 18.18 7.05 313 2 0 0 12.5318.18 6.98 315 none 1.12 0 12.53 18.18 6.97 307 6 0 3.83 12.53 18.186.97 312 21  1.12 3.83 12.53 18.18 7.01 315 4 0 4.59 12.53 18.18 7.00320 2 1.12 4.59 12.53 18.18 7.02 318 2 0 0 0 36.35 6.97 330 none 1.12 00 36.35 6.97 342 none 0 3.83 0 36.35 7.00 348 none 1.12 3.83 0 36.356.98 347 none 0 4.59 0 36.35 6.98 343 6 1.12 4.59 0 36.35 7.00 346 4 0 06.83 36.35 7.04 331 none 1.12 0 6.83 36.35 7.01 357 none 0 3.83 6.8336.35 6.97 349 none 1.12 3.83 6.83 36.35 7.02 351 none 0 4.59 6.83 36.357.05 349 4 1.12 4.59 6.83 36.35 7.02 360 4 0 0 12.53 36.35 6.98 315 none1.12 0 12.53 36.35 6.94 344 none 0 3.83 12.53 36.35 6.99 350 none 1.123.83 12.53 36.35 7.00 348 none 0 4.59 12.53 36.35 6.98 357 4 1.12 4.5912.53 36.35 6.99 354 4

FIGS. 3A and 3B and Table 6 highlight some aspects of the data. First,high levels of cystine, cysteine, or tyrosine increased precipitation.However, addition of the highest concentration of sodium pyruvate tested(36.36 mM) inhibited precipitation associated with cystine or cysteineand most of the concentrations of tyrosine tested, whereas lowerconcentrations of pyruvate tested were not as effective in stabilizingmedia. Addition of 9.09 or 18.18 mM pyruvate stabilized some samples ascompared to samples containing 4.53 mM pyruvate. However, even 36.35 mMsodium pyruvate did not stabilize media containing 4.59 mM tyrosine.

1. A method for stabilizing a concentrated feed medium for feeding amammalian cell culture comprising including in the feed medium at leastabout 9 mM pyruvate, wherein the feed medium comprises cysteine and/orcystine, wherein the sum of the concentrations of cysteine and/orcystine is at least about 7.9 mM, wherein the pH of the feed medium isfrom about 5.8 to about 7.4, wherein the feed medium comprises tyrosineand the tyrosine concentration is not more than about 4.4 mM, andwherein the feed medium with the included pyruvate is stable for atleast about 1 week at room temperature.
 2. The method of claim 1,comprising including in the feed medium at least about 18 mM pyruvate.3. The method of claim 2, comprising including in the feed medium atleast about 30 mM pyruvate.
 4. The method of claim 1, wherein the pH ofthe feed medium is from about 6.0 to about 7.2.
 5. The method of claim1, wherein the feed medium comprises at least about 6.0 mM cysteine. 6.The method of claim 5, wherein the feed medium comprises at least about12.0 mM cysteine.
 7. The method of claim 6, wherein the feed mediumcomprises not more than about 40 mM cysteine.
 8. The method of claim 1,wherein the feed medium comprises at least about 3 mM tyrosine.
 9. Themethod of claim 8, wherein the feed medium comprises at least about 4 mMtyrosine.
 10. The method of claim 1, wherein the feed medium is serumfree.
 11. The method of claim 1, wherein the feed medium comprises aprotein hydrolysate.
 12. The method of claim 1, wherein the feed mediumhas an osmolarity from about 200 mOsm to about 1000 mOsm.
 13. The methodof claim 12, wherein the feed medium has an osmolarity from about 500mOsm to about 1000 mOsm.
 14. The method of claim 1, wherein the feedmedium comprises at least about 0.5 mM cystine.
 15. The method of claim14, wherein the feed medium comprises at least about 1.0 mM cystine. 16.The method of claim 4, wherein at least about 30 to about 40 mM sodiumpyruvate is included in the feed medium, wherein the feed mediumcomprises from about 3 mM to about 4 mM tyrosine, wherein the feedmedium is stable for at least about 2 weeks at room temperature, whereinthe feed medium does not comprise cystine, and wherein the feed mediumcomprises at least about 12 mM cysteine.
 17. A feed medium for amammalian cell culture comprising at least about 9 mM pyruvate and atleast about 5 mM cysteine, wherein the feed medium may or may notcomprise cystine, wherein the sum of the concentrations of cysteine andcystine is at least about 7.9 mM, wherein the pH of the feed medium isfrom about 5.8 to about 7.4, wherein the feed medium comprises tyrosineand the tyrosine concentration is not more than about 4.4 mM, andwherein the medium is stable for at least about 1 week at roomtemperature.
 18. The feed medium of claim 17, wherein the pH of the feedmedium is from about 6.0 to about 7.2.
 19. The feed medium of claim 17,comprising at least about 18 mM pyruvate.
 20. The feed medium of claim19, comprising at least about 30 mM pyruvate.
 21. The feed medium ofclaim 17, comprising at least about 12.0 mM cysteine.
 22. The feedmedium of claim 21, comprising at least about 16.0 mM cysteine.
 23. Thefeed medium of claim 17, wherein the feed medium comprises not more thanabout 40 mM cysteine.
 24. The feed medium of claim 17, comprising atleast about 3 mM tyrosine.
 25. The feed medium of claim 24, comprisingat least about 4 mM tyrosine.
 26. The feed medium of claim 17, whereinthe feed medium is serum free.
 27. The feed medium of claim 17,comprising a protein hydrolysate.
 28. The feed medium of claim 17,wherein the feed medium is chemically defined.
 29. The feed medium ofclaim 17, wherein the feed medium has an osmolarity from about 200 mOsmto about 1000 mOsm.
 30. The feed medium of claim 29, wherein the feedmedium has an osmolarity from about 500 mOsm to about 1000 mOsm.
 31. Thefeed medium of claim 17, wherein the mammalian cell culture is a CHOcell culture, and wherein the pyruvate is sodium pyruvate.
 32. The feedmedium of claim 17, comprising at least about 0.5 mM cystine.
 33. Thefeed medium of claim 32, comprising at least about 1.0 mM cystine. 34.The feed medium of claim 17, comprising (a) from about 3 mM to about 4mM tyrosine, (b) from about 30 mM to about 40 mM sodium pyruvate, and(c) at least about 12 mM cysteine and not more than about 20 mMcysteine, wherein the feed medium is stable for at least about 2 weeksat room temperature, wherein the pH of the feed medium is from about 6.0to about 7.4, and wherein the feed medium does not comprise cystine. 35.A method for producing a protein comprising culturing mammalian cellsthat produce the protein in a base medium, feeding the culture with thefeed medium of claim 17, and recovering the protein from the culturemedium.